Spatial molecular analysis of tissue

ABSTRACT

Various methods and devices for spatial molecular analysis from tissue is provided. For example, a method of spatially mapping a tissue sample is provided with a microarray having a plurality of wells, wherein adjacent wells are separated by a shearing surface; overlaying said microarray with a tissue sample; applying a deformable substrate to an upper surface of said tissue sample; applying a force to the deformable substrate, thereby forcing underlying tissue sample into the plurality of wells; shearing the tissue sample along the shearing surface into a plurality of tissue sample islands, with each unique tissue sample island positioned in a unique well; and imaging or quantifying said plurality of tissue sample islands, thereby generating a spatial map of said tissue sample. The imaging and/or quantifying may use a nucleic acid amplification technique.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 62/404,825, filed Oct. 6, 2016, which is herebyincorporated in its entirety to the extent not inconsistent herewith.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with United Statesgovernmental support awarded by National Science Foundation award no.SBC 5710003967, United States Department of Agriculture award nos.8000063044, 80000069085 and 8000074077 and National Institutes of Healthaward no. MGH-APF 087126. The United States Government has certainrights in this invention.

REFERENCE TO A SEQUENCE LISTING

A sequence listing containing SEQ. ID. NOs. 1-44 is submitted herewithand is specifically incorporated by reference.

BACKGROUND OF INVENTION

The spatial localization of gene expression can unravel importantinsights into tissue heterogeneity, functionality and pathologicaltransformations, but the ability to maintain this spatial informationremains an enduring challenge in tissue sections routinely used forpathology. Amplification-based spatial gene expression analysis methodsprovide good sensitivity and specificity but decouple the analyteisolation and biochemical detection steps, making them low throughputand laborious constraints, limiting the translation of the above methodsinto routine research and clinical practice. Direct probe-basedhybridization techniques such as single molecule FISH allow directvisualization of single RNA molecules in their native cellular contextbut are not amenable on tissue sections in a high throughout manner. Inaddition, off-target binding of FISH probes and cellularauto-fluorescence can also become a limiting factor in imaging tissuesamples. Methods to perform spatially-mapped transcriptome analysis on atissue section can identify multiple targets simultaneously but theymust trade-off between the histologic reference and the quality ofrecovered biomaterials as staining and manual identification are oftenneeded.

The limitations described above are addressed herein by a specialplatform to reliably pixelate a tissue section into separate islands oftissue that reside in separate wells and that can be individuallyanalyzed, thereby providing a highly sensitive, reproducible andefficient platform for spatial analysis of tissue. The methods andsystems are compatible with on-chip picoliter real-time reversetranscriptase loop mediated isothermal amplification (RT-LAMP) reactionson a histological tissue section, including without any analytepurification, while preserving the native spatial location of thenucleic acid molecules. In an exemplary methodology, the entire processfrom tissue loading on microchip to results from RT-LAMP, can be carriedout in less than two hours. This technique with its ease of use, fastturnaround, and quantitative molecular outputs, is invaluable for arange of applications, including tissue analysis, for researchers andclinicians.

SUMMARY OF THE INVENTION

The methods and systems provided herein overcome conventionallimitations and problems associated with spatial imaging of tissuesamples, including spatial gene expression useful for tissuecharacterization. The ability to reliably and efficiently achievespatial molecular analysis of tissue relies on pixelating tissue samplesinto individual wells. Corresponding efficient processing of thepixelated tissue, including by reliable interaction of reagent materialsand tissue, bulk fluid application and removal, and avoiding cross-talkbetween different wells containing different pixelated tissue, togetherensure the methods and systems provide significant functional benefitsthat ensure reliable spatial mapping of a tissue sample.

Provided herein are various methods of spatially mapping a tissuesample. The method is particular suited for obtaining information abouta biological tissue that may spatially vary, such as arising fromdifferent cell type, cell state, pathogen, disease state, therapeuticresponse state, target analyte, including presence or absence of atarget nucleic acid. The method may comprise the steps of: providing amicroarray having a plurality of wells, wherein adjacent wells areseparated by a shearing surface; overlaying the microarray with a tissuesample; applying a deformable substrate to an upper surface of thetissue sample; applying a force to said deformable substrate, therebyforcing underlying tissue sample into the plurality of wells; shearingthe tissue sample along the shearing surface into a plurality of tissuesample islands, with each unique tissue sample island positioned in aunique well; and imaging or quantifying the plurality of tissue sampleislands, thereby generating a spatial map of the tissue sample. Thespatial map may be observed in real time on a display and/or may bedigitally recorded for later analysis.

The method may further comprise the step of pre-spotting or printing oneor more molecules on a surface of the plurality of wells. The moleculesmay be useful in the imaging or quantifying step. For example, inapplications where nucleic acid amplification occurs, the molecules maycomprise enzymes and/or primers useful in the amplification technique.

Any of the methods may further comprise the step of removing thedeformable substrate before the imagining or quantifying step andapplying a reagent for use in the imaging or quantifying step. In thisaspect, some materials may be pre-spotted/printed and other materialsmay be applied at a later time point in the method.

The reagent may comprise a plurality of reagents for nucleic acidamplification, the method further comprising the step of amplifying eachof said plurality of wells using a nucleic acid amplification technique,including polymerase chain reaction (PCR) or an isothermal technique,thereby generating a plurality of amplified products. Accordingly, theimaging may comprise analyzing the plurality of amplified products,thereby generating a spatial gene analysis of the tissue sample. Theimaging may be optical in nature, such as by fluorescence orphase-contrast microscopy. The imaging may be electrical in nature, suchas by monitoring a change in an electrical parameter in the wells,including using a FET, such as an ISFET.

The step of applying a force upon the microarray may be by any techniquethat reliable forces the deformable substrate into the plurality ofwells, such that the tissue is sheared into separate pieces (e.g.,“islands” or “pixelated”), with each piece in a unique well. Suitableforce application techniques include by spinning the assembledmicroarray, tissue sample and deformable substrate in a centrifuge. Theresultant centrifugal force accordingly forces the deformable substrate,and corresponding underlying tissue, into the wells. Similarly, anon-centrifugal uniform force may be applied over the deformablesubstrate, such as a weighted block or driver that results in desireddeformable substrate deformation into the wells and correspondingshearing of the tissue sample into corresponding wells.

The wells may be described as having a volume of less than or equal to1000 pL; a cross-sectional dimension of less than or equal to 1 mm, or amaximum depth of less than or equal to 1 mm.

The method is compatible with a range of tissue samples, including ahistological tissue section. The tissue sample may be described ashaving an average thickness, including of less than or equal to 20 μm ora range between 3 μm and 20 μm. The tissue sample may be cryopreserved.

The deformable layer may comprise a polymer or an elastomer, or anymaterial that exhibits a deformation property such that after theapplied force is removed, the deformable layer exits the wells andrelaxes back to a rest state. In contrast, the plurality of tissuesample islands remains within the wells. The wells may be coated with anadhesion-promoting layer that ensures a bonding force between the tissueand the well that is greater than the adhesion force between the tissueand the deformable layer. This ensures that tissue islands remain in thewells even when the deformable layer exits the wells. Accordingly, thedeformable layer may be coated with an anti-adhesion-promoting layer tominimize the adhesive force between the tissue and the deformablesubstrate.

The deformable substrate may be formed of a polymer that ispolymethylsiloxane (PDMS), SU-8, polyethylene glycol (PEG), aphotoresist, a PEG-based polymer or any combination thereof.

The method may further comprise the step of delivering one or morereagents and/or molecules to the plurality of wells before the step ofoverlaying said microarray with the tissue sample, wherein the one ormore reagents and/or molecules are useful for the imaging or quantifyingstep.

The method may further comprise the step of delivering one or morereagents and/or molecules to the plurality of wells after the shearingstep, wherein the one or more reagents and/or molecules are useful forthe imaging or quantifying step and the delivering is by one or morethan one delivery application steps.

The method may further comprise the step of processing the tissue sampleislands by: removing the deformable substrate; applying reagents used toimage and/or quantify the tissue sample islands to each of the wells,wherein the applying step comprises: covering the wells with liquidreagent, wherein the liquid reagent enters the wells by capillaryaction; immersing the wells with liquid reagent in an inert coveringfluid having a density that is less than the liquid reagent density,thereby enveloping each well containing a tissue sample island andliquid reagent without entering the wells; and removing excess reagentby forcing a gas over the microarray, thereby avoiding cross-talkbetween different wells. In this manner, the liquid filling is rapid,reliable, and avoids unwanted material communication between adjacentwells. The covering fluid may comprise mineral oil.

Any of the tissue sample islands may be fixed and permeabilized,including to facilitate desired interaction between biological materialand reagents and/or molecules in the liquid reagent.

The nucleic acid amplification technique may comprise PCR or anisothermal technique, such as reverse transcription, loop-mediatedisothermal amplification (RT-LAMP).

Any of the methods may comprise fluorescent imaging to facilitatemapping of the tissue sample that has been pixelated into tissueislands.

The method may further comprise adding an optically detectable dye orparticle to each of the plurality of wells.

The mapping may be a quantifiable mapping, such as by measuring anoptical, electrical and/or mechanical parameter in each of the wells.Mechanical properties may include stress-induced mechanical bending orresonant frequency of a mechanical resonator (e.g. a quartz crystalmicrobalance or MEMS cantilever). Electrical parameters may be measured,for example, by field effect transistors.

The methods provided herein may are compatible for a range ofapplications, including for one or more of: an on-chip spatial geneexpression analysis; on-chip spatial RNA sequence analysis; on-chipspatial methylation analysis; on-chip gene mutation analysis; copynumber variation analysis; or insertion and deletion analysis.

The method may be for pathogen detection, tissue functionalityassessment, or pathological diagnostics. Examples of pathogen detectioninclude detection of bacteria, fungi, mold, or viruses, including byamplification of target nucleic acids specific for the genome of a rangeof bacteria or viruses. The configuration of systems and methodsprovided herein allows for highly multiplexed detection, includingdifferent target analytes having, for example, different fluorescentspectrum.

The method is compatible with a range of well numbers, including greaterthan 500 wells up to 10,000, 100,000 or 1×10⁶. In this manner, even forrelatively large surface area tissue, a desired spatial resolution maybe maintained.

Also provided herein are devices for performing any of the methodsdescribed herein. For example, provided is a device for generating apixelized tissue sample comprising: a substrate; a plurality of wellssupported by or embedded in the substrate; and a shearing surfacepositioned between adjacent wells, wherein the shearing surface has asharp edge configured to sever a tissue sample under an appliedcentrifugal force into a plurality of tissue sample islands, with eachwell containing a unique tissue sample island so as to maintain spatialinformation of a tissue sample during use.

The substrate may be silicon, a glass, a metal, an insulator or adielectric.

The shearing surface may be described as having a sharp edge along whichduring use the tissue sample is sheared. The well may have a geometricshape that is inverted pyramidal. Other geometric shapes are compatible,so long as the sharp edge is accessible to the tissue and the tissue iscapable of being forced into the well.

Each of the wells may comprise a target-specific primer set and anenzyme for nucleic acid amplification. The primer set is selected, asknown in the art, for specificity to a desired portion of a nucleicacid, such as in a genome indicative of a pathogen or mutation.

The device may further comprise a deformable substrate configured tocover the plurality of wells and during application of a force, to forcea tissue sample into each of the plurality of wells.

Also provided herein is a method for generating a pixelated,spatially-preserved tissue sample. Numerous functional advantages areachieved with such methods, including the ability to rapidly, reliablyand at a high sensitivity and resolution, characterize molecularvariation of a tissue sample, The method may comprise the steps of:providing a microarray having a plurality of wells, wherein at least aportion of each edge of the well is a shearing surface; providing atissue sample in contact with each of the wells; overlaying a deformablelayer on said tissue sample; applying a force upon the deformable layer,thereby forcing the deformable layer and the tissue sample into theplurality of wells and shearing the tissue sample into a plurality oftissue sample islands positioned in the plurality of wells; and relaxingthe force, thereby removing the deformable layer from the plurality ofwells, while maintaining the plurality of tissue sample islandspositioned in the plurality of wells, thereby generating a pixelated,spatially-preserved tissue sample.

Also provided is a method of determining spatial gene expression by:loading a cryopreserved tissue sample onto a chip, the chip comprising asubstrate having a plurality of inverted pyramidal microwells withsharp, defined edges; placing an organic polymer on top of thecryopreserved tissue sample; centrifuging the substrate to force theorganic polymer to force the tissue sample into the microwells, therebyshearing the tissue sample and forming a pixelated tissue sample;removing the organic polymer from the pixelated tissue sample; applyinga plurality of PCR (or LAMP) reagents in bulk by pipetting the reagentsto cover the entirety of the microwells and allowing the reagents toenter the microwells by capillary action; applying mineral oil to coverthe microwells and applying forced air at an angle to remove excessreagents while keeping the reagents located inside the microwells;performing PCR (or LAMP) in the microwells by incubating the chip at adesired temperature to create a plurality of PCR products; and analyzingthe presence of the plurality of PCR products.

The well size may be about 5 μm on a side to about 1000 μm on a side.The well size is about 1 μm deep to about 1000 μm deep. Accordingly, thewells may be described a microwell, referring to at least one dimensionthat is less than 1 mm.

The primers may be printed or spotted in the wells prior to the transferand pixelation of the tissue.

The organic polymer may be described as pliable and capable of enteringthe microwells and pushing the tissue sample into the well whencentrifugal force is applied, and bending back to its original shape andvacating the microwells when centrifugal force is no longer applied.

The organic polymer may be polydimethylsiloxane (PDMS), SU8,photoresist, and any PEG based material.

Any of the methods described herein may have a plurality of PCR productsthat are fluorescent, thereby facilitating imaging and, if desired,quantifying.

The nucleic acid amplification reagents may be suitable for reversetranscription loop-mediated isothermal amplification (RT-LAMP) or PCR.

The presence of the plurality of PCR or LAMP products can be detected byfluorescence or electrical means, including by fluorescent imaging orFET devices, including ISFETs. Mechanical properties may be measuredusing one or more mechanical sensors, such as QCM (quartz crystalmicrobalance) or MEMS (microelectromechanical system) resonator.

Also provided is a gene expression analysis chip comprising a substratecomprising a plurality of microwells having inverted pyramidal walls andsharp, distinct edges. The substrate may be silicon oxide on silicon.The size of the microwells may be about 5 μm on a side to about 1000 μmon a side and/or about 1 μm deep to about 1000 μm deep.

The chip can be used to analyze many different cell populations.

Also provided is a kit for performing spatial gene expression analysison tissue, the kit comprising at least one of the any chips describedherein, at least one polymerase enzyme, and dinucleotide triphosphates(dNTPs) in a single, dry format; wherein said reagent preparation iswater soluble and stable above 4° C.

The kit may further comprise a target-specific primer set.

The kit may further comprise a positive control.

The polymerase and target-specific primer set may be printed or spottedonto the chip.

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

The features, objects and advantages other than those set forth hereinwill become more readily apparent when consideration is given to thedetailed description below. Such detailed description makes reference tothe following drawings, wherein:

FIG. 1. Exemplary overall process flow schematic (top). LNCaP cells areinjected into a mouse and prostate cancer xenograft obtained (1);Xenograft is immediately frozen after embedding in OCT (2); A 7 umtissue cryosection is loaded onto chip (3) and the tissue is “pixelated”and transferred into individual wells (4); Post pixelation, the tissueis fixed with acetone and treated with proteinase K (5); Picolitervolume RT-LAMP reagents are loaded onto the chip through a rapid bulkloading technique (6); Quantitative gene expression is visualizedthrough real-time imaging of the amplification reaction in each well.Tissue pixelation process schematic (bottom blue box). A PDMS loaded ontop of tissue-chip assembly (3 a), the PDMS shears the tissue at sharpwell edges and pushes into wells under centripetal force in a standardcentrifuge. The tissue adheres to the silanized (APTES) well surfacesand the PDMS is removed. Reagent bulk loading process schematic (bottomred box). RT-LAMP reagents are pipetted on chip in bulk (5 ul) (5 a) andcompressed air is blown on it at an angle. (5 b). Excess reagents areremoved and fluid only inside wells is retained due to capillary forces.

FIG. 2A. SEM and DAPI-fluorescence characterization of the same chipafter tissue pixelation. Tissue partitioning and division into smallpixels (pixelation) can be clearly visualized as tissue seen inside thewells. FIG. 2B. Chip characterization after bulk picoliter volumereagent loading in wells. Rhodamine dye was filled in wells forcharacterization. Well edges can be seen as dark lines showing that theyare above the fluid level and there is no overflow between adjacentwells. Partially filled wells indicated by a lower fluorescence were asmall fraction of total wells on chip and confined to the chipboundaries.

FIG. 3A. Standard curve for cells spiked in reaction. The standard curveshows a good linear fit. A single cell could be reliably detected. FIG.3B. Raw thermocycler amplification curves for cells spiked in reaction.The standard curve shows a good linear fit. A single cell could bereliably detected.

FIGS. 4A-4D. On-chip RT-LAMP. FIG. 4A. Raw fluorescence images ofreal-time RT-LAMP with tissue on chip at four different time points.FIG. 4B. Fluorescence bar graphs of the raw images showing adifferential increase in fluorescence over time. The gain influorescence over time is calculated taking time=0 image (initial) asthe reference. FIG. 4C. Spatial threshold analysis showing the spatiallymapped threshold times. Note that the tissue boundaries are maintainedduring reaction. Threshold time=0 refers to blanks. FIG. 4D. Rawamplification curves of a row showing positive and negative wells.

FIGS. 5A-5D. On-chip RT-LAMP: Cancer vs non-cancer control. FIG. 5A. Rawfluorescence images of real-time RT-LAMP with prostate cancer tissue onright and non-cancer (mouse skeletal muscle) tissue on left of chip atfour different time points. FIG. 5B. Fluorescence bar graphs of the rawimages showing a differential increase in fluorescence over time. Thegain in fluorescence over time is calculated taking time=0 image(initial) as the reference. Note the amplification occurring only forthe cancerous tissue. FIG. 5C. Spatial threshold analysis showing thespatially mapped threshold times. Threshold time=0 refers to blanks.FIG. 5D. Raw amplification curves of a row showing positive and negativewells. Note that well 23 shows no amplification and is captured in thethreshold analysis.

FIGS. 6A-6D. Chip characterization. FIG. 6A. Optical image of the chip.FIG. 6B. SEM image of the wells. FIG. 6C. SEM image of the sharp welledge shown as red box in FIG. 6B. Note that the edge width is close to 1micron. FIG. 6D. Surface profilometer measurement of fabricated siliconoxide micro wells showing the depth of the wells.

FIGS. 7A-7B. Bulk reagent loading characterization. FIG. 7A. Stitchedfluorescent image of the complete chip showing filling distribution ofthe wells using a Rhodamine dye. It can be seen that only some of thewells at the edges are partially filled. The well edges are dark andclearly visible indicating no cross-talk between adjacent wells. FIG.7B. Histogram showing the well fluorescence distribution after filling.Note that lower fluorescence is attributed to partially filled wells andhigher fluorescence values are for wells without tissue.

FIGS. 8A-8B. Raw thermocycler fluorescence data (FIG. 8A) and standardcurve (FIG. 8B) of RT-LAMP reaction for TOP2A with purified total RNAextracted from LNCaP cell. 10⁴ cells had 37.6 ng/ul concentration ofpurified total RNA per reaction as measured nanodrop spectrophotometer.A good linear fit was observed in the standard curve and total RNA from1 cell equivalent could be detected.

FIGS. 9A-9B. Raw thermocycler fluorescence data (FIG. 9A) and standardcurve (FIG. 9B) of RT-LAMP reaction for TOP2A with purified total RNAextracted from tissue. 1× has 98 ng/ul concentration of purified totalRNA per reaction. A good linear fit was observed in the standard curve.

FIGS. 10A-10B. Raw thermocycler fluorescence data (FIG. 10A) andstandard curve (FIG. 10B) of RT-PCR reaction for TOP2A with purifiedtotal RNA extracted from LNCaP cell. 10⁴ cells had 37.6 ng/ulconcentration of purified total RNA per reaction as measured nanodropspectrophotometer (same as for RT-LAMP reactions). In contrast with theRT-LAMP reaction, the RT-PCR reaction could only detect total RNA from a100 cells equivalent. The blanks start to amplify after 30 cycles forthis reaction. This shows the superiority our designed RT-LAMP assay forTOP2A over the existing RT-PCR assay.

FIG. 11. 4 point parameter model used for sigmoidal fitting of the rawamplification curves. The equation for the sigmoidal fit is given in thered box inside the figure and the corresponding parameters arerepresented in the data fit shown as an example. The threshold time wastaken as (yo+0.2*a) which is in line with our thermocycler thresholdtime calculation.

FIG. 12A. Curve fitting analysis showed for a positive and a negativewell. FIG. 12B. Amplification fluorescence curves for all wells.

FIG. 13A. Raw fluorescence image at time 0 showing regions with andwithout tissue. FIG. 13B. Processed image with numbered wells (FIG.13C). FIG. 13D. Raw amplification curves for marked regions showing thatthe positive wells (with tissue) amplify while the adjacent negativesdon't and that the tissue boundary remains preserved duringamplification. This confirms that there is no cross talk betweenadjacent wells.

FIG. 14A. Curve fitting analysis showed for a positive and a negativewell. FIG. 14B. Amplification fluorescence curves for all wells.

FIG. 15A. Raw fluorescence image at time 0 showing regions with andwithout tissue. FIG. 15B. Processed image with numbered wells (FIG.15C). FIG. 15D. Raw amplification curves for marked regions showing thatthe positive wells (with cancerous tissue) amplify while the negatives(with non-cancerous tissue) don't and also that the tissue boundaryremains the tissue boundary remains preserved during amplification. Thisconfirms that our on-chip reaction is specific.

FIGS. 16A-16B. No primer on-chip negative control. FIG. 16A. Rawfluorescence images of RT-LAMP reaction on chip with no-primers in thereaction mix. FIG. 16B. Raw fluorescence curves of all the wells showingno amplification.

FIGS. 17A-17B. RNase treated-on-chip negative control. FIG. 17A. Rawfluorescence images of RT-LAMP reaction on chip with RNase A treatedtissue. FIG. 17B. Raw fluorescence curves of all the wells showing noamplification.

FIGS. 18A-18D. On-chip RT-LAMP 300 um wells. FIG. 18A. Raw fluorescenceimages of real-time RT-LAMP with tissue on chip at four different timepoints. FIG. 18B. Fluorescence bar graphs of the raw images showing adifferential increase in fluorescence over time. The gain influorescence over time is calculated taking time=0 image (initial) asthe reference. FIG. 18C. Spatial threshold analysis showing thespatially mapped threshold times. Note that the tissue boundaries aremaintained during reaction. Threshold time=0 refers to blanks. FIG. 18D.Amplification curves for all wells after curve fitting.

FIGS. 19A-19D. On-chip RT-LAMP 300 um wells. FIG. 19A. Raw fluorescenceimage at time 0 showing regions with and without tissue. FIG. 19B.Processed image with numbered wells (FIG. 19C). FIG. 19D. Rawamplification curves for marked regions showing that the positive wells(with tissue) amplify while the negatives (without tissue) don't andalso that the tissue boundary remains preserved during amplification.

FIGS. 20A-20D. On-chip RT-LAMP 500 um wells. FIG. 20A. Raw fluorescenceimages of real-time RT-LAMP with tissue on chip at four different timepoints. FIG. 20B. Fluorescence bar graphs of the raw images showing adifferential increase in fluorescence over time. The gain influorescence over time is calculated taking time=0 image (initial) asthe reference. FIG. 20C. Spatial threshold analysis showing thespatially mapped threshold times. Note that the tissue boundaries aremaintained during reaction. Threshold time=0 refers to blanks. FIG. 20D.Amplification curves for all wells after curve fitting.

FIGS. 21A-21D. On-chip RT-LAMP 500 um wells. FIG. 21A. Raw fluorescenceimage at time 0 showing regions with and without tissue. FIG. 21B.Processed image with numbered wells (FIG. 21C). FIG. 21D. Rawamplification curves for marked regions showing that the positive wells(with tissue) amplify while the negatives (without tissue) don't andalso that the tissue boundary remains preserved during amplification.

FIGS. 22A-22K. Overall process flow schematic. FIG. 22A. LNCaP cells areinjected into a mouse and prostate cancer xenograft obtained. FIG. 22B.Xenograft is resected and immediately frozen and embedded in optimalcutting temperature compound (OCT). FIG. 22C. A 7 um tissue cryosectionis loaded onto our microchip. FIG. 22D. A cured PDMS block is loaded ontop of tissue-chip assembly. FIGS. 22E-22F. The PDMS shears andpartitions the tissue into small pixels at sharp well edges and pushesthem into wells under centripetal force in a standard centrifuge. Thepixelated tissue adheres to the silanized (APTES) well surfaces and thePDMS is removed. We call this process “Tissue pixelation” (Time=2minutes). FIG. 22G. Post pixelation, the tissue is fixed with acetone(Time=10 minutes). A proteinase K digestion is performed after this tocreate a pathway for amplification enzymes to reach the target nucleicacids inside cells. (Time=30 minutes). FIG. 22H. RT-LAMP reagents arepipetted on chip in bulk (5 ul). FIG. 22I. Compressed air is blown on itat an angle inside mineral oil. FIG. 22J. Excess reagents are shearedaway and fluid only inside wells is retained due to capillary forces. Inthe above steps, picoliter volume RT-LAMP reagents (˜175 pL/well) areloaded onto the chip through a rapid instrument-free technique we call“bulk picoliter reagent loading”. (Time=2 minutes). FIG. 22K.Quantitative gene expression is visualized through real-time imaging ofthe amplification reaction in each well performed using only a hot plateat 65 C and a fluorescence microscope. (Time=45 minutes).

FIGS. 23A-23C. Off-chip RT-LAMP assay characterization. FIG. 23A.Amplification curves and standard curve of the TOP2A mRNA RT-LAMP withpurified total RNA extracted from LNCaP cells. 10⁴ cells had 940 ng ofpurified total RNA per reaction as measured with nanodropspectrophotometer. FIG. 23B. Amplification curves and standard curve ofthe RT-PCR assay for TOP2A mRNA performed using previously publishedprimers²¹. Our RT-LAMP assay can detect TOP2A mRNA from a single cell inreaction tube, whereas the RT-PCR assay can detect mRNA from only up to100 cells (˜9.4 ng total RNA) in a reaction tube (25 ul per reaction).The amounts of RNA per reaction for each dilution was the same as inRT-LAMP (FIG. 23A) to allow direct comparison. FIG. 23C. Amplificationcurves and standard curve of the TOP2A mRNA RT-LAMP assay with wholecells spiked directly into the reaction tubes. TOP2A down to a singlecell could be reliable amplified.

FIGS. 24A-24H. Tissue pixelation and Bulk picoliter reagent loadingcharacterization. FIGS. 24A-24D. SEM characterization after tissuepixelation. Tissue partitioning and division into small pixels can beclearly visualized as tissue seen inside the wells. The blue box in FIG.24A is shown in FIG. 24B and the blue box in FIG. 24B is shown in FIG.24C and FIG. 24D. FIGS. 24E-24F. DAPI-fluorescence imaging of the samepixelated tissue showing nuclei inside the well boundaries. FIG. 24Fshows the region in yellow box in FIG. 24E. FIGS. 24G-24H.Characterization after bulk picoliter reagent loading in tissue loadedwells. Fluorescent rhodamine dye was filled in the wells forcharacterization of cross-over across wells. FIG. 24G shows the lowmagnification image of dye filled tissue (*) and no-tissue (**) regionsand FIG. 24H shows the high magnification image of a dye filled region(shown in yellow box in FIG. 24G) with tissue. Well edges are seen asdark lines showing that they are above the fluid level and there is nooverflow between adjacent wells. Partially filled wells indicated by alower fluorescence were a small fraction of total wells on chip andconfined to the chip boundaries as shown in FIG. 7A.

FIGS. 25A-25E. On-chip RT-LAMP: Cancer vs non-cancer control. FIG. 25A.Raw fluorescence images of real-time RT-LAMP with prostate cancer tissueon right and non-cancer (mouse skeletal muscle) tissue on left of chipat four different time points (*Non-cancer, **Cancer). FIG. 25B.Fluorescence bar graphs of the raw images showing a differentialincrease in fluorescence over time. The gain in fluorescence over timeis calculated taking time=0 image (initial) as the reference. Note theamplification occurring only for the cancerous tissue. FIG. 25C. Spatialthreshold analysis showing the spatially mapped threshold times.Threshold time=0 refers to blanks. FIG. 25D. Raw amplification curves ofa row showing positive and negative wells. FIG. 25E. Fluorescence curvesfor all wells after curve fitting.

FIGS. 26A-26B. On-chip RT-LAMP with mRNA FISH on serial sections. FIG.26A and FIG. 26B show two sets of serial sections. For each set, on-chipRT-LAMP is performed on section 1 (1-2) and mRNA FISH is performed onsection 2 (3-4). 1. Baseline-subtracted fluorescence images of real-timeRT-LAMP with tissue on chip at three different time points showing theincrease in fluorescence over time. 2. Spatial threshold analysisshowing the spatially mapped threshold times. Threshold time=0 refers towells which are not amplifying. 3. DAPI (blue) and TOP2A mRNA FISH (red)images of the consecutive section showing spatial heterogeneity in TOP2AmRNA expression. 4. Pixelated intensity map of mRNA FISH fluorescence.The spatial pattern of TOP2A expression is similar between the two assaytypes.

FIG. 27. Optical image of the chip beside a quarter. The dark region inthe chip is the array of microwells.

FIGS. 28A-28E. SEM characterization of rat heart tissue pixelation.Tissue partitioning and division into small pixels can be clearlyvisualized as tissue inside the wells. The blue box in FIG. 28A is shownin FIG. 28B and the blue box in FIG. 28C is shown in FIG. 28D. FIG. 28Eshows pixelated tissue inside a single well.

FIGS. 29A-29E. Regional Image Analysis for FIG. 24. FIG. 29A. Rawfluorescence image at time=0 showing regions with and without tissue.FIG. 29B. Zoomed in processed image with numbered wells. The inset showsthe entire processed image. FIGS. 29C-29D. Raw amplification curves formarked regions (blue and green) showing that the positive wells (withtissue) amplify while the adjacent negative wells do not. The tissueboundary remains preserved during the amplification reaction confirmingthat there is no cross talk between adjacent wells. FIG. 29E.Representative amplification curve with sigmoidal fit from a positiveand negative well.

FIGS. 30A-30D. Specificity validation of TOP2A mRNA FISH in culturedcell lines. Fluorescence micrographs show nuclear stain (Hoechst; toprow) and TOP2A mRNA FISH (Quasar 647; bottom row). FIGS. 30A-30B.TOP2A-negative mouse 3T3 fibroblasts and RAW 264.7 macrophages show nosignificant TOP2A mRNA FISH signal. FIGS. 30C-30D. TOP2A-positive humanprostate cancer cell lines PC-3 and LNCaP show significant TOP2A mRNAFISH signal. The scale bar on the bottom right of each image is 25micrometers.

FIGS. 31A-31B. Illustrate an embodiment of a device for the pixelationof a tissue sample.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

“Spatially mapping” is used broadly herein to refer to obtaining usefulinformation about a tissue in a manner that is spatially preserved.Accordingly, the information may correspond to molecular informationthat spatially varies. In contrast, certain conventional assays simplyprovide a “reading”, including presence/absence or magnitude from atissue sample. The ability to read-out a signal in a spatially-preservedmanner provides access to a number of useful applications, includingspatial mRNA analysis of tissue, pathogen detection localization, andinformation that normally is associated with histological staining ofproteins or only morphology of cells, including localization of mutated(e.g., cancerous) cells with attendant tumor shape characteristics andspreading information. Spatial mapping may be used to detect or readnucleic acid molecules and genes. Accordingly, the spatial mappingmethods and devices provided herein provide a useful platform for cellsand pathogen detection/analysis, tissue research, 3-D modeling of geneexpression, and is readily compatible with any amplification methods,including nucleic acid amplification. Such gene mapping and analysis canoccur in relatively short run-times, such as less than about 2 hours.

“Array” refers to an ordered placement of wells to provide the desiredpixilation of tissue into a plurality of tissue islands. Each tissueisland can then undergo individual and simultaneous processing, such asfor nucleic acid amplification. Such amplification accordingly occurs ina manner that is spatially preserved. Conventional amplificationtechniques, in contrast, generally occur in a manner that spatialinformation is lost. Accordingly, as used herein “well” broadly refersto a volume in which tissue can be confined, including in a manner toensure there is no or minimal cross-talk between wells. That is, tissueor target analyte does not significantly pass between wells. Suchunwanted cross-talk would adversely impact spatial information andspatial sensitivity.

As used herein, “substrate” refers to a material, layer or otherstructure having a surface, such as a receiving surface, supporting oneor more components or devices including an array or microarray. Arraysmay be embedded in substrates so that the array is formed within andmade the same material as the substrate. Arrays embedded in substratemay be manufactured from a single piece of material. Substrates whichmay be useful in the methods and devices described herein includesilicon, glasses, metals, insulators and/or dielectrics. Substrates maybe composite materials. The substrate and/or supported array may also bereferred herein as a chip.

“Deformable substrates” are substrates having sufficient elasticity suchthat they deform under an applied force and relax back to or nearlytheir undeformed shape upon removal of the applied force. Materialsuseful as deformable substrates include polymers, for example, PDMS,SU-8, PEG, and photoresists.

“Array” refers to material or device having a number of wells, receivingchambers, void spaces or is otherwise configured to hold a number oftissue samples. Microarrays refer to wells that have at least dimensionthat is less than 1 mm. An array may have any number of wells and may beprovided in various configurations including a grid, as describedherein. Wells useful in the described arrays may have any geometricshape including inverted pyramids, cones, and rounded bottom wells withcircular, square or polygonal cross-sections. Arrays may be described interms of one or more dimensions (e.g. depth, width), volume and/orshape. Microarrays may have greater than or equal to 1000 individualwells, greater than or equal to 2500 wells, or optionally, greater thanor equal to 5000 wells, depending on the desired spatial resolution.“Spatial resolution”, accordingly, is directly correlated to the spacingbetween wells and the well footprint size. As wells are more tightlyspaced, the spatial resolution increases. Any of the methods and devicesprovided herein, may have a spatial resolution as high as 1 μm, 10 μm or100 μm. Spatial resolution refers to the minimum distance at whichreliable differences arising from tissue-differences within the tissuesample are detectable. Depending on the application of interest, thespatial resolution is correspondingly tailored, with relative large wellfootprint surface areas and volumes for those applications not requiringhigh spatial-sensitivity and where large tissue sample volumes aredesired. In contrast, for applications where detailed differences oversmall regions are desired, the array may have relatively small surfacearea footprint and/or volume, so fine differences over short distancesare detectable.

“Shearing surface” is a surface positioned between two or more of thewells that is capable of severing, cutting or otherwise shearing atissue sample into more than one piece when the tissue sample is forcedinto the shearing surface. Shearing surfaces may be configured as toform the walls, edges or boundaries of the wells of a microarray.Shearing surfaces may be defined as their cross-sectional width at ornear the point of contact with a tissue sample. For example, shearingsurfaces may have a width of less than or equal to 5 μm, or optionally,less than or equal to 2.5 μm. The shearing surface may also be describedas having a relatively high slope and appropriate depth, both to ensurethe tissue is reliably cut or sheared and that the cut or sheared tissueremains within the well and physically separated from adjacent wells.For example, the shearing surface may correspond to a well edge having adepth of between about 20 μm and 200 μm and an average slope of betweenabout 0.5 and 1.5, or about 1, with the top of the well meeting atsharp-edged region where the slope changes from a positive to negativedirection, such as over a sharp-edged distance that is less than orequal 5 μm, toward a sharp-point where the slope changes from a positiveto negative direction over a distance that is less than 0.1 μm.Conceptually, the shearing surface may be continuous so as to sever thetissue sample into separate islands, with one unique island per well.

“Imaging” and/or “quantifying” is used broadly herein to refer to anymethod of analyzing a target analyte or biomarker, depending on theapplication of interest. For example, the amount or evenpresence/absence of a target nucleic acid in a tissue sample island.Imaging may utilize dyes, including fluorescent dies, or fluorescentlylabeled tags, with any imaging device such as a camera, electricaldevice or computer to quantitatively (e.g. measuring intensity) orqualitatively determine the amount of nucleic acid in a sample. Imagingand/or quantifying includes electrically-based techniques, such asimplementing FETs and/or ISFETs, to measure nucleic acid presence,quantity or concentration.

“Polymerase chain reaction” (PCR) is a commonly used technique thatenzymatically replicates targeted portions of nucleic acids which usesthermal cycling for example to denature, extend and anneal the nucleicacids to amplify the amount of a nucleic acid sample analyzed by takingthe sample through 3 temperature steps. These steps are for theannealing of the primer (lowest temperature), extension (the actualamplification, medium temperature) and denaturation of the product,which make up one cycle of the PCR. In each cycle the amount of nucleicacid is amplified twice the value before the cycle. By cycling manytimes, the nucleic acid at hand can be amplified orders of magnitude.Relevant measures include measurement of a PCR by-product, such as pHchanges or hydrogen ion levels as hydrogen ions are generated asbyproducts of the amplification reaction. For more specificamplification assessment, the measurement may relate to generatedpyrophosphates whose generation is electrically detected, or a detectionof the amplified DNA sequence, such as by a binding event to a surfacethat is electrically detected by the corresponding FET. See, e.g., U.S.Pat. Nos. 8,945,912, 9,433,943, incorporated specifically by referencedfor the FET-based detection, including ISFET.

“Inert covering fluid” refers to layer of fluid selected to cover thearray of wells and facilitates removal of excess fluid reagents,including by forcing air over the wells at a sufficient force to removethe excess liquids on top the wells, while the liquids in the wellremain under a relatively higher capillary force or surface tension inthe relatively small-dimensioned well.

EXAMPLE 1 RT-LAMP Analysis of Prostate Cancer

Described herein is an on-chip spatial gene expression analysistechnique that can perform real-time nucleic acid amplification,including reverse transcriptase loop mediated isothermal amplification(RT-LAMP) starting from tissue samples, while keeping the native spatiallocation of the nucleic acid preserved. We engineered a silicon oxidechip with an array of microwells that serve as independent picolitervolume RT-LAMP reaction vessels. The wells were designed to haveknife-like sharp edges (referred herein as a “shearing surface”) thathelp in tissue partitioning-and-transfer into wells starting from atissue cryosection. A capillary action based reagent loading techniquewas developed to fill all the wells on the chip simultaneously whilepreventing reagent overflow between wells in the final loaded chip.Using this platform we amplified the TOP2A mRNA starting from a 7 microntissue section of a prostate cancer xenograft and visualized thevariation in amplification threshold times across the tissue.

The example described herein eliminates all of the drawbacks describedabove by use of a tissue cryosection as a starting tissue sample,requires minimal sample processing and performs parallel picoliterreverse transcription loop-mediated isothermal amplification (RT-LAMP)reactions in an array of wells (volume˜175 pL) with tissue in them. Thenative spatial distribution of nucleic acid in tissue is preservedthroughout the process.

RT-LAMP: Loop-mediated isothermal amplification (LAMP) overcomes thedependence on expensive equipment (via elimination of thermocycling andthe requirement for machine-based result detection) while amplifying DNAor RNA rapidly and specifically. Notomi et al., Nucl. Acids Res. 28:E63(2000); U.S. Pat. No. 6,410,278. Because of the advantage in rapid,efficient, and specific amplification of small amounts of DNA and RNA,LAMP has emerged as a powerful tool to facilitate genetic testing forthe rapid diagnosis of viral and bacterial infectious diseases inclinical laboratories. A novel nucleic acid amplification method, knownas reverse transcription loop-mediated isothermal amplification(RT-LAMP), has been recently used for detection influenza A virus,Newcastle disease virus, classical swine fever virus and porcinereproductive and respiratory syndrome virus (Notomi et al, Nucleic AcidsRes. 2000 Jun. 15; 28(12):E63; Pham et al., J Clin Microbiol. 2005April; 43(4):1646-50; Chen et al., Mol Cell Probes. 2009 April;23(2):71-4.)

The substrate can be any material, including, but not limited to,silicon glass, metal, insulator, and dielectrics.

The chip or microarray can be used for analyzing different sample typessuch as cells, and for differentiating and quantifying different cellpopulations on a chip based on their genetic markers/make-up.

The sensing modality of the PCR products can include, but is not limitedto, fluorescent, mechanical and electrical. For example, the techniquecan be combined with field effect transistors (FETs) and theamplification reaction can be detected electrically via a change in pHor a mechanical signal from QCM or MEMS cantilever.

The described methods and devices can also be coupled with histologicalor immunostaining prior to amplification and thus a protein levelanalysis is possible in addition to the nucleic acid analysis.

LAMP is an exemplary amplification reaction from tissue that is robustagainst inhibitors such as cellular debris or blood which usuallyinhibit a PCR reaction. LAMP uses 4-6 primers which identify 6-8 regionson the template for amplification which makes it more specific than PCR.

Moreover, LAMP is isothermal so it only needs a portable heater to carryout the reaction, eliminating the bulky instruments required for PCR. Wedesigned fingernail sized silicon oxide—on-silicon chips with an arrayof 5625 inverted pyramidal wells having knife-like sharp distinct edgesto carry out the reactions. Once the tissue is loaded onto our chip, itis partitioned and transferred inside the wells in a process we call“tissue pixelation”. This is followed by tissue fixation,permeabilization, loading of wells with amplification reagents and finalRT-LAMP reaction on-chip carried out on a hot plate. (FIG. 1).

We select prostate cancer tissue for this example. Despite being themost common cancer diagnosed in men and second leading cause of cancerdeath in the United States, accounting for more than 25,000 deathsannually according to 2015 cancer statistics [49], the molecularmechanisms fueling the prostate cancer pathogenesis remain relativelyunknown. Topoisomerase II alpha (TOP2A), a nuclear enzyme involved inprocesses such as chromosome condensation and chromatid separation, hasbeen shown to be upregulated with increasing Gleason score and withhormone insensitivity in prostate carcinoma [50]. The combination ofprostate cancer xenografts grown in mice using LNCaP cell line and TOP2AmRNA were chosen to visualize the spatial mRNA variation within thexenograft tissue using our technique. With a rapid turn-around-time of 2hours, starting from sample acquisition to RT-LAMP reaction, our lowcost technique can perform spatially mapped nucleic acid amplificationtest (NAAT) in any basic laboratory with minimal facilities.

TOP2A mRNA RT-LAMP off-chip: Here, we characterize a sensitive andspecific RT-LAMP reaction for TOP2A mRNA. Provided is a novel RT-LAMPreaction for amplifying TOP2A mRNA using 6 sequence specific primers.The details about the primer design are described herein, with specificsequences provided in the tables.

As a first characterization of the reaction, the off-chip (‘tube-based’)TOP2A RT-LAMP reaction is performed using purified total RNA from cellsand tissue sections (FIGS. 13A-13D and FIGS. 14A-14B). The standardcurve in both the cases showed a good linear fit (R²=0.93 and 0.98 fortotal RNA from cells and tissue respectively). TOP2A from purified totalRNA of 1 cell equivalent could be detected by our designed RT-LAMPreaction. We compared this reaction sensitivity with RT-PCR byperforming reactions with the same RNA concentrations and previouslypublished RT-PCR primers for TOP2A. As can be seen from FIGS. 10A-10B,the RT-PCR reaction was 2 orders of magnitude less sensitive and couldonly detect TOP2A from purified RNA of 100 cells equivalent. The nextstep was to test the robustness of our RT-LAMP reaction. We spiked 1 to100 LNCaP cells directly in a 25 ul tube-based reaction and tested foramplification. (FIGS. 3A-3B). The figures show the amplificationfluorescence curves and standard curve for 1, 10, 50 and 100 cellsspiked in reaction. The reaction with a single cell could be reliablyamplified. Together these data demonstrate the sensitivity, specificityand robustness of the RT-LAMP reaction for TOP2A mRNA.

Tissue pixelation and bulk reagent loading: To perform the RT-LAMPreaction on chip from tissue sample there are two unique preparatorysteps required: 1. Tissue pixelation—Dividing the tissue cryosection onchip into small separated bits/pixels that are put into theircorresponding underlying wells for downstream parallel and independentamplification reactions. 2. Bulk picoliter reagent loading—Loading theamplification reagents into the wells post tissue pixelation. The wellshave a volume of ˜175 pL and there are 5625 wells on a chip which needto be filled with reagents while making sure there is no overflowbetween any two wells. The schematic in FIG. 1 shows the tissuepixelation protocol. When the PDMS block is placed on top of the tissueand the whole assembly is exposed to centripetal force in a standardcentrifuge, the flexible PDMS pushes its way into the wells, shearingthe tissue at the well edges in the process. The tissue sticks to thepre-silanized (APTES) chip surface while the PDMS restores to itsoriginal shape in the absence of the force. We named this process“tissue pixelation” as a continuous cryosection disc (7 um thick) isdivided into thousands of small pixels in this step. FIG. 2A shows theDAPI stained and SEM images of the tissue in wells after pixelation. Thewell edges can be clearly visualized as dark lines in the DAPI stainedfluorescent images. These data show that the tissue is completely insidethe wells after the pixelation step and the tissue partitioning intopixels is complete.

FIG. 1 panel (3) shows the bulk picoliter reagent loading protocol.After the reagents are pipetted in bulk (<5 uL), the whole chip (withtissue inside wells and pipetted reagents on top) is immediatelyimmersed in mineral oil and compressed air is blown on it. With themineral oil acting as an envelope for the reagents, the excess ofreagents are sheared away due to air pressure while the capillary forcesretain fluid only inside the wells. In this step, we filled 7225 wellswith ˜175 pL volume per well. There are a few commercial solutions forspotting arrays of nanodroplets but none of these can spot picolitervolumes in such close spacing. These commercial systems also have largedead volumes (milliliters of solution that are used to fill reservoirsbut can't be used for droplets) and loading over 5000 wells using anysuch commercial micro-injector system would take hours. The bulk loadingtechnique we showed here can be scaled to fill larger arrays withmillions of wells using the same principle in a matter of 1-2 minutes.We used fluorescent rhodamine dye to characterize this process as shownin FIG. 2B. The figure shows the fluorescent images after filling thewells with the dye. The well edges are above the fluid level and can beseen as dark lines showing that there is no cross-talk between adjacentwells. Partially filled wells can be seen with lower fluorescenceintensity and were seen only near the chip boundaries. FIGS. 7A and 7Bshow a histogram of well fluorescence distribution and complete chipdata for fluorescence.

On-chip real time RT-LAMP reaction: To perform the real-time on chipRT-LAMP reaction we first fixed the pixelated tissue using acetone.Fixing the tissue in acetone takes only about 10 mins and stops the RNAdegradation at room temperature. Acetone is a precipitative fixative andhas been shown to provide good RNA yields for downstream amplificationreactions. The tissue was then treated with proteinase K (5-10 mg/ml)for 30 mins. The pre-treatment of tissues with proteinase K digests theproteins in the cell membrane and makes them permeable to polymerase andreverse transcriptase enzymes. This allows the RT-LAMP reagents topenetrate the cells and carry out the amplification reaction. Both theabove steps are standard in many biological protocols such as for insitu hybridization, or in situ PCR.

After the above steps are performed, the tissue/chip is ready for theon-chip RT-LAMP reaction. The reagents were loaded using the previouslydescribed bulk reagent loading technique and the amplification reactionwas carried out on a hot plate at 65 C, imaged every 2 mins using anOlympus BX51 fluorescence microscope. The on-chip amplification reactionwas completed in 35 mins and the progressive product accumulation ineach well was visualized as proportional increase in the fluorescence inthe corresponding well. These real-time fluorescence curves were used tocalculate the threshold times for each well. FIGS. 4A-4D show the rawfluorescence images at different time points, the differential spatialfluorescence bar graphs and the spatial threshold time analysis. Asigmoidal curve fitting was performed on the raw fluorescence curves andthreshold time was calculated as shown in FIG. 11. FIGS. 12A-12B showthe curve fitting parameters for this experiment and all amplificationcurves. Analyzing regions close to the tissue boundary showed that therewas no crosstalk between adjacent wells (FIGS. 13A-13D). The rawamplification curves were found highly comparable to the ones obtainedoff-chip using the commercial thermocycler.

To ensure that the signal observed was not due to spurious/non-specificamplification, we performed a series of on-chip controls. In the firstnegative control, primers were omitted from the reaction mixture and noamplification was observed in the tissue (FIGS. 16A-16B). To test if thereaction is not amplifying genomic DNA, usually a no RT control isperformed. Since BST polymerase itself has significant RT activity weperformed an RNase digestion step using 100 ug/ml RNase A for 60 minsprior to amplification to degrade all the RNA. No amplification wasobserved in this case (FIGS. 17A-17B). As a final test for thespecificity of the reaction on chip, cancer and non-cancer (mouseskeletal muscle tissue) tissue were loaded on the same chip and thereaction was performed. FIGS. 5A-5D show that only the cancerous tissueamplified validating the specificity of our on-chip assay. As thefield-of-view reduces for higher magnification objectives, we could onlyimage 784 wells for all the real-time on-chip measurements.

We harnessed the sensitivity, specificity and robustness of a LAMPreaction and combined that with basic micro fabrication techniques todeliver a technique that is simple and easy to perform, has a rapidoverall run time of less than 2 hours and has the ability to quantitate.The process, which currently requires only a fluorescent microscope anda hot plate to carry out can be easily integrated into a completelyportable setup using a smartphone and in-built heater making thetechnique accessible to even labs without a microscope. We demonstratedthe sensitivity of the LAMP reaction off-chip for TOP2A down to a singlecell spiked in tube while the theoretical sensitivity of the LAMPreaction goes down to a single molecule. The fluorescence curves foron-chip experiments look comparable to the off-chip thermocyclerfluorescence curves. This is the first demonstration of on-chippicoliter RT-LAMP reactions with tissue in them.

The size of the chip and well are both variable and can be designed andfabricated depending on the number of samples to be analyzedsimultaneously on a chip and the resolution required. We demonstratedon-chip amplification for 300 and 500 um well sizes apart from thestandard 100 um well size. (FIGS. 18A-18D, 19A-19D, 20A-20D and21A-21D). With the advent of multiplexing in LAMP, internal control(housekeeping gene) can be incorporated in each well and the geneexpression data can be normalized accordingly for a more accurateinterpretation. The functional benefit of the instantly describedtechnique addresses a number of problems associated with conventionalprior techniques, as summarized in Table 1.

TABLE 1 Comparison to prior techniques. Prior Technique Problems In situLow sensitivity, long run times hybridization In situ PCRNon-quantitative, long run times, poor reproducibility Laser CaptureLong sample acquisition times, Microdissection purification times,resource intensive, followed by only small regions analyzed at a timeqPCR

EXAMPLE 2 Pixelated Spatial Gene Expression Analysis from Tissue

Described herein is a rapid technique that performs on-chip picoliterreal-time reverse transcriptase loop mediated isothermal amplification(RT-LAMP) reactions on a histological tissue section without any analytepurification while maintaining the native spatial location of thenucleic acid molecules. This example includes a method of amplifyingTOP2A messenger RNA (mRNA) in a prostate cancer xenograft with 100 μmspatial resolution and by visualizing the variation in threshold timesacross the tissue. The on-chip reaction was validated throughfluorescence mRNA in situ hybridization (ISH). The entire process, fromtissue loading on microchip to results from RT-LAMP can be carried outin less than two hours. This technique with its ease of use, fastturnaround, and quantitative outputs is an invaluable tissue analysistool for researchers and clinicians in the biomedical arena.

The spatial localization of gene expression can unravel importantinsights into tissue heterogeneity, functionality and pathologicaltransformations, but the ability to maintain this spatial informationremains an enduring challenge in tissue sections routinely used forpathology. Amplification-based spatial gene expression analysis methodsprovide good sensitivity and specificity but decouple the analyteisolation and biochemical detection steps, making them low throughputand laborious¹⁻³. Direct probe-based hybridization techniques such assingle molecule FISH allow direct visualization of single RNA moleculesin their native cellular context but off-target binding of FISH probesand cellular auto-fluorescence become a limiting factor in imagingtissue samples⁴⁻⁷. Methods performing spatially-mapped transcriptomeanalysis on a tissue section can identify multiple targetssimultaneously but they must trade-off between the histologic referenceand the quality of recovered biomaterials as staining and manualidentification are often needed⁸. Also, since they utilizeRNA-sequencing platforms, it makes them very resource intensive andexpensive to perform⁸⁻¹¹. These constraints limit the translation of theabove methods into routine research and clinical practice.

Here, we introduce a technique which improves upon these drawbacks byanalyzing a starting sample of tissue cryosection and performingparallel picoliter RT-LAMP reactions with minimal sample processing.LAMP is an isothermal reaction which has been shown to be robust againstinhibitors in tissue that inhibit a PCR reaction¹². It uses 4-6 primerswhich identify 6-8 regions on the template for amplification which makesit more specific than PCR¹³. We designed fingernail-sized siliconoxide-on-silicon chips with an array of 5,625 inverted pyramidal ˜175 pLwells having knife-like sharp distinct edges to carry out the reactions(FIGS. 27 and 6A-6C). Once a tissue cryosection is loaded onto our chip,it is partitioned and transferred inside the wells in a process we call“tissue pixilation,” also referred herein as “tissue islands.” Thistissue pixilation process which divides a solid tissue section intosmall tissue pixels takes less than 2 minutes. This is followed bytissue fixation (10 minutes), permeabilization (30 minutes), loading ofwells with amplification reagents (2 minutes), and finally on-chipRT-LAMP reaction on a hot plate (45 minutes) (FIGS. 22A-22K). The nativespatial distribution of nucleic acid in tissue is preserved throughoutthe process.

This example utilizes frozen sections of human prostate tissuexenografts grown in mice. Prostate cancer is the most commonly-diagnosedcancer in men and is the second leading cause of cancer death in men inthe United States, accounting for more than 25,000 deaths in 2015¹⁴. Themolecular mechanisms fueling prostate cancer pathogenesis remainrelatively unknown¹⁵⁻¹⁹; however, topoisomerase II alpha (TOP2A), anuclear enzyme involved in chromosome condensation and chromatidseparation, has been shown to be upregulated with increasing Gleasonscore and with hormone insensitivity in prostate carcinoma²⁰. LNCaPxenografts grown in mice was chosen to visualize the spatial variationof TOP2A mRNA using our technique. With a rapid turn-around time of lessthan 2 hours, starting from sample acquisition to RT-LAMP reaction, ourlow-cost technique can perform spatially mapped nucleic acidamplification testing (NAAT) in a typical analytical laboratory.

TOP2A mRNA RT-LAMP in a thermocycler: The first step is to develop andcharacterize a sensitive and specific RT-LAMP reaction for TOP2A mRNA.We designed a novel RT-LAMP reaction for amplifying TOP2A mRNA using 6sequence specific primers (primer sequences provided in Table 2).

To characterize our TOP2A reaction, RT-LAMP experiments using purifiedtotal RNA from xenograft tissue sections and cultured LNCaP cells wereperformed in a commercial thermocycler and compared with RT-PCRreactions performed using previously published primers with same RNAconcentrations²¹. FIG. 23A and FIGS. 9A-9B show the amplification curvesand the standard curve for RT-LAMP reactions from purified RNA fromLNCaP cells and tissue xenograft, respectively. A good linear fit forthe standard curves was obtained for both the reactions (R²=0.93 and0.98 for total RNA from cells and tissue respectively). FIG. 23B showsthe similar amplification curves and standard curve for TOP2A qRT-PCRreaction. The total amounts of RNA per reaction for each dilution wasidentical in the RT-LAMP and RT-PCR reactions for comparison. TOP2ART-LAMP was found to be at least one order of magnitude more sensitivethan the corresponding RT-PCR reaction with detection down to a singlecell equivalent of total RNA (in 25 μL tube-based reactions).

Next, to demonstrate the robustness of our RT-LAMP reaction againstinhibitors, using hemocyotemeter counting and serial dilutions we spiked1 to 100 LNCaP cells directly in a 25 μL reaction tube and performedRT-LAMP in a thermocycler. FIG. 23C shows the amplification fluorescencecurves and standard curve for this experiment. Reaction with a singlecell could be reliably amplified even in the presence of cell lysate. Asseen from FIG. 23A and FIG. 23C, the amplification reaction works betterfor 100 cells spiked directly into the reaction as compared to purifiedRNA from 100 cells. We believe that this is due to the loss of some RNAduring the RNA purification process. We expect the on-chip sensitivityto go down to a single molecule as the on-chip reaction volumes will be5 orders of magnitude lower in microchip wells (Vol˜175 pL) making theanalyte concentrations equivalently higher when compared to tube basedthermocycler reactions (Vol˜25 μL). As shown in the on-chip experimentson Prostate cancer xenograft tissue, the tissue debris/chassis remainsattached to the bottom of the wells and does not become an effectivepart of the reaction in the solution above. This causes the effect oftissue contaminants on the reaction in wells to be minimized.

Tissue pixelation and bulk picoliter reagent loading: To perform theRT-LAMP reaction on a microchip from tissue samples, we developed twounique preparatory steps: (A) Tissue pixelation—A continuous tissuecryosection disc (7 um thick) was partitioned into thousands of smalltissue “pixels” and placed into the corresponding microwells. This wasdone by pushing a deformable substrate, such as a flexible curedpolydimethylsiloxane (PDMS) block into the wells with overlaying tissueusing centrifugation in a standard centrifuge (1 min@1500 g force). Whenthe flexible PDMS pushes its way into the wells under centrifugalforces, it shears and partitions the tissue at the sharp well edges. Thetissue sticks to the pre-silanized (APTES) well surface and the PDMSlayer restores back to its original shape in the absence of force asshown in the FIGS. 22D-22F. Characterization of this process usingscanning electron microscopy (SEM) and fluorescence imaging for Nucleiafter DAPI staining is shown in FIGS. 24A-24F. The well edges can beclearly visualized as dark lines in the DAPI stained fluorescent images.These data show that the tissue is completely inside the wells after thepixelation step and the tissue partitioning into pixels is complete,allowing for independent RT-LAMP reaction from tissue in each picowell.The 2-D tissue distribution is maintained throughout the process. FIGS.28A-28E show additional SEM images of tissue pixelation for a rat hearttissue section.

(B) Bulk picoliter reagent loading—To fill the tissue loaded wells with˜175 pL of reagents per well, we developed a capillary action-basedinstrument-free loading technique. 5 μL of reagents were pipetted on thechip and then the chip was immediately immersed in mineral oil. Mineraloil has a lower density than water based reaction-mixture and hence itstays on top of the reagent-filled wells. With mineral oil acting as anenvelope, excess reagents were sheared away using air pressure whilecapillary forces retained fluid only inside the wells. The process wascharacterized using fluorescent rhodamine dye. FIGS. 24G-24H show welledges seen as dark lines indicating they are above the fluid level andthat there is no cross-talk between adjacent wells. 98.2% of the wellswere found to be fully filled (fluorescence intensity greater than 22a.u). and partially filled wells d were found only near the chipborders. FIGS. 7A-7B show the complete chip data for this process. Theabove two processes ensure independent picoliter RT-LAMP reactions ineach well starting from a tissue sample. Mineral oil is one example ofan “inert covering fluid” that does not substantively react with any ofthe relevant materials and is used to facilitate bulk fluid handling andremoval.

Real-time microchip RT-LAMP: Before performing the real-time microchipRT-LAMP reaction, the pixelated tissue was first fixed using acetone for10 minutes to prevent RNA degradation at room temperature²². Followingthis, the tissue was treated with proteinase K (7.5 mg/ml for 30 mins),which digests the cell membrane proteins making the cells permeable topolymerase and reverse transcriptase enzymes.³ This allows RT-LAMPreagents to penetrate the cells and carry out amplification. As opposedto lysing the whole tissue, in which scenario the tissue debris wouldhave been completely mixed with the overlying solution in wells, usingproteinase K digestion to expose the target analyte inside the tissueprevents the tissue contaminants from becoming an effective part of thereaction. The amplification reagents were loaded using the previouslydescribed technique and the amplification was performed on a hot plateat 65 C. Imaging was done every 2 minutes using a 5× objective and TRITCfilter in an Olympus BX51 fluorescence microscope. The on-chipamplification reaction was completed in 35 minutes and the progressiveproduct accumulation in each well was visualized as proportionalincrease in the fluorescence in the corresponding well. These real-timefluorescence curves were used to calculate the threshold times for eachwell. FIGS. 4A-4C show the fluorescence images at different time points,the differential spatial fluorescence bar graphs and the spatialthreshold time analysis showing the variation in threshold times,respectively. FIG. 12A shows the raw fluorescence data over time for arow of wells. A sigmoidal curve fitting was performed on the rawfluorescence curves from all the imaged wells and threshold time wascalculated as shown in FIGS. 11 and 29E. Fluorescence curves from allthe imaged wells are shown in FIG. 12B after the fitting analysis.Analyzing regions close to the tissue boundary showed that the tissueboundary was maintained during the reaction suggesting that there was nocrosstalk between adjacent microwells and our rapid tissue pixelationand reagent loading technique indeed isolated each picowell. (FIGS.4A-4C and FIG. 12A). The regions without any tissue showed nonon-specific amplification. To demonstrate the scalability of ourtechnology, we also performed similar on-chip reactions from tissue ontwo different well sizes of 300 um and 500 um, the results for which areshown in FIGS. 18A-18D, 19B-19D, 20A-20D and 21B-21D. To further ensurethat the signal observed was not due to spurious amplification, twonegative controls, one with no primers in the reaction mix and otherwith RNase A (100 μg/ml for 1 hr) treated tissue for RNA degradationwere performed and no amplification was observed for either. (FIGS.16A-16B and 17A-17B).

As a final specificity test of our on-chip assay, we challenged ourplatform by loading cancer and non-cancer (mouse skeletal muscle tissue)tissue on the same chip and performing the reaction simultaneously onthem. FIGS. 25A-25E show the fluorescence images at different timepoints, the differential spatial fluorescence bar graphs and the spatialthreshold time analysis showing the variation in threshold times,respectively. FIG. 25D shows the raw fluorescence data over time for arow of wells and fluorescence curves from all the imaged wells are shownin FIG. 25E after the fitting analysis. It is observed that only thecancerous tissue amplified validating the specificity of our on-chipassay. We imaged at most 784 wells (100 um well size) for our real-timeon-chip measurements.

Comparison with mRNA fluorescence in situ hybridization (FISH): Asfurther validation of our technique, we performed mRNA FISH andmicrochip spatial amplification on serial sections of the tissue. FISHprobes for human TOP2A mRNA were confirmed to specifically stain humanprostate cancer cells (PC3, LNCaP) and to not stain non-expressing mousecell lines (3T3, RAW 264.7) (FIGS. 30A-30D). The probe sequences areprovided in Table 3. FIGS. 26A-26B show two sets of experiments forwhich microchip amplification and mRNA FISH were performed onconsecutive tissue sections. For microchip amplification, FIG. 26A panel1 and FIG. 26B panel 1 show baseline subtracted fluorescence images atthree different time points of the real-time amplification,demonstrating an increase in fluorescence over time. FIG. 26A panel 2and FIG. 26B panel 2 show the spatial threshold time analysis,demonstrating the variation in threshold times across the tissuesections for the two sets. FIG. 26A panel 3 and FIG. 26B panel 3 showconfocal fluorescence micrographs of nuclei (blue) and TOP2A mRNA FISH(red), showing spatial heterogeneity in TOP2A mRNA expression. Thesemicrographs were then pixelated at the same spatial resolution as thespatial threshold time maps and displayed in FIG. 26A panel 4 and FIG.26B panel 4. A substantial correlation in the general spatial pattern isapparent between the results through the two TOP2A mRNA measurementmethods. However, because higher mRNA expression levels yield fasteramplification, mRNA FISH fluorescence intensity is negatively correlatedwith threshold time. Spearman correlation coefficient values between thepixelated maps of mRNA FISH and spatial thresholds were −0.56(p=8.7881e−55) and −0.57 (p=3.9886e−54) for the data sets in FIGS. 26Aand 26B, respectively. These correlations were substantially strongerthan those comparing pixelated maps of nuclear stains and spatialthresholds, with correlation values of −0.38 (p=9.0580e−24) and −0.37(p=9.9159e−22), respectively.

The methods and devices described herein can be tuned to performquantitative spatially-mapped nucleic acid microchip analysis of anytissue sample type on a simple hot plate and fluorescence microscope. Itcan also be integrated into a completely portable setup using asmartphone and in-built heater making the technique accessible even tolabs without a microscope¹². These methods and devices allow analysis ofsmall-to-large tissue regions without any cross-talk between individualtissue pixels. The tissue pixelation and bulk picoliter reagent loadingtechniques perform sample partitioning of solid-tissue samples andliquid-reagent samples respectively, in easy-to-perform steps that takeonly 1-2 minutes. This is remarkable considering that commercialsolutions for spotting arrays of nanodroplets cannot spot picoliters ofvolume in close spacing, have large dead volumes (non-usable reagentpool) and suffer from long sample loading times (loading 5000 wellswould take several hours)^(28,29). Our technique can be scaled to filllarger arrays with millions of wells using the same principle in amatter of minutes. Both the size of our chip and wells can be tuned tomeet sample size and spatial resolution requirements and we demonstratethis by showing on-chip amplification for 300 and 500 μm wells apartfrom the 100 μm wells. We expect the same should be possible for lowerwell sizes that go down to a single cell per well, in which case we canmake the wells deeper to get similar reaction volumes as for the 100 umwells to keep the same reaction kinetics. The described methods anddevices, which can be easily translated into routine analysis inresource-starved settings to highly sophisticated measurements, has manyimportant clinical and biological applications such as understandingtumor evolution and heterogeneity, predicting patient outcomes,post-operative characterization of surgical margins and detecting pointmutations across the tissue³⁰.

Cells and Xenografts. LNCaP prostate cancer cells were obtained from andverified by the American Type Culture Collection (ATCC). The cells werecultured per the recommended standard in a 37° C. humidified incubatorwith 5% CO2 atmosphere.

LNCaP subcutaneous Xenografts were grown in immunocompromised nude mice(Jackson Laboratory). LNCAP cells were first incubated and grown toconfluence. Then, these confluent cells were suspended in 10% matrigelat a concentration of 2×10⁷ cells/60 μL and 60 μL of this matrigel-cellsolution was injected subcutaneously into both flanks of the animal. Themice were then monitored daily for the presence of tumors. As soon astumors were visible, volume measurements were taken using digitalcalipers twice a week, and tumor volumes were calculated using theformula Volume=(Length*Width{circumflex over ( )}2)/2. Once tumorsreached 50,000 mm{circumflex over ( )}3 in volume, the mice weresacrificed by overdosing with isofluorane anesthesia, after which thetumors were immediately excised and divided to be placed in optimalcutting temperature compound (OCT), or 4% PFA.

Primer Sequences: All primer sequences for the RT-LAMP and RT-PCRreactions were synthesized by Integrated DNA Technology (IDT) and arelisted in Table 2. Primerexplorerv4 was used for designing the RT-LAMPprimers for TOP2A mRNA. The cDNA sequence for TOP2A was obtained fromNCBI database using NCBI Reference Sequence: NM_001067.3.

Off-chip reactions. RT-LAMP assay was designed to target theTopoisomerase II alpha (TOP2A) mRNA. The RT-LAMP assay comprised of thefollowing components: 1× final concentration of the isothermalamplification buffer (New England Biolabs), 1.4 mmol/L each ofdeoxy-ribonucleoside triphosphates (dNTPs), 10 mmol/L of MgSO4 (NewEngland Biolabs), and 0.4 mol/L of Betaine (Sigma-Aldrich). Thesecomponents were prepared in bulk and stored at −20° C. betweenexperiments. In addition to the buffer components, 1 μL of primer mixconsisting of 0.2 μM of F3 and B3, 1.6 μM FIP and BIP, and 0.8 μM ofLoopF and LoopB, 0.64 U/μL Bst 2.0 WarmStart DNA Polymerase (New EnglandBiolabs), 0.08 U/μL AMV reverse transcriptase (New England Biolabs), and1× EvaGreen (Biotium), a double-stranded DNA (dsDNA) intercalating dye,was included in the reaction. 10 μL template of the appropriateconcentration and 1.92 μL of DEPC-treated water (Invitrogen) was addedto make the final reaction volume 25 μL.

The RT-PCR reaction was carried out using the RNA UltraSense™ One-StepQuantitative RT-PCR System (Thermofisher) according the manufacturer'sinstructions. A 50 μL reaction mix contained 2.5 μl of RNA UltraSense™Enzyme Mix, 10 μl of RNA UltraSense™ 5× Reaction Mix™, 1 μl of 10 μMForward primer, 1 μl 10 10 μM backward primer, 1 μl of SYBR Green dye(Thermofisher), and 34.5 μl of template of appropriate startingconcentration.

Template for the RT-LAMP reactions included either RNA extracted fromLNCaP cells or LNCaP prostate xenograft tissues, or whole LNCaP cellsspiked in the reaction mix. All RNA extractions were performed using theRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.All RT-LAMP and RT-PCR reactions consisted of non-template negativecontrols, the amplification of which indicated a contaminated test.

All the off-chip LAMP tests were carried out in 0.2 mL PCR reactiontubes in an Eppendorf Mastercycler® realplex Real-Time PCR System. Thetubes were incubated at 60° C. for 60 minutes in the thermocycler, andfluorescent data was recorded every 1 minute. The off-chip PCR testswere conducted on the same thermocycler but with the following recipe:RT incubation at 50° C. for 50 minutes, 2 minutes DNA denaturation at95° C., and 50 cycles of thermocycling from 95° C. (15 seconds) to 60°C. (30 seconds). Fluorescence data was recorded after each cycles of thereaction.

Chip Fabrication and Chip Silanization. Undoped silicon wafers(University Wafers) were piranha cleaned for 10 minutes and a 160 nm ofsilicon oxide was thermally grown in a furnace at 1150° C. for 90minutes. A 2 μm layer of positive photoresist AZ1518 (AZ ElectronicMaterials) was spin-coated on the unpolished side of the wafer and wassoft-bake on a hot plate at 110° C. for 8 minutes. The same process wasrepeated on the polished size of the wafer. The photoresist on the shinyside was patterned using an EVG 620 aligner with a high resolutiontransparency mask (FineLine Imaging). The wafer was then developed inAZ400K (AZ Electronics) to remove the exposed regions for 1 minute. Theunprotected silicon oxide was etched in 10:1 buffered oxide etchant(VWR) to reveal the underlying bare silicon. This was followed bystripping the wafer of photoresist in a Remover PG (MicroChem) at 70° C.for 30 minutes. The wafer was then anisotrpically etched in a TMAH bath(1:1 TMAH:DI) for 100 mins at 80 C to etch inverted square pyramidalwells with sharp well edges. To passivate the chip, a 125 nm of siliconoxide was thermally grown in a furnace at 1150° C. for 90 minutes.

To render a positive charge on well surfaces for tissue adhesion,silicon chips were silanized with functional groups using(3-Aminopropyl)triethoxysilane (APTES). The chips were dipped in a glassjar containing 0.2% APTES for 60 seconds. The chips were then dipped 5times in a separate vessel containing distilled water. This step wasrepeated three more times with the water being replaced between eachstep. The silanized chips were stored in a desiccator, and were usedwithin 15 days of silanization.

Tissue Pixelation, Fixation, and Proteinase K digestion: The frozentissue was cryosectioned at a thickness of 7 μm onto the center of themicrochip and stored at −20° C. Once ready for use, the tissue was takenout of the −20° C. freezer and dried immediately to minimize RNaseactivity within the tissue. A short heating step at 105° C. for 5seconds was incorporated to ensure that the tissue is stabilized on thechip. A clean block of PDMS was then placed on top of the tissue and thewhole PDMS-chip conjugate was centrifuged at 3000 rpm for 1 minute topress the tissue into the wells in a process known as tissue pixelation.The block of PDMS was discarded, and a second heating step at 105° C.for 7 seconds was carried out to ensure the tissue is firmly adhered andstable on the chip.

A standard acetone fixation protocol was followed to fix the tissue ontothe chip. The chip was placed on a small glass petri dish filled withacetone and incubated at −20° C. for 8-9 minutes. After the incubationstep, the chip underwent a series of wash steps. First, half of theacetone was discarded from the petri dish and an equal amount of coldPBS (Fisher Scientific) was poured into the petri dish to replace theacetone. The petri dish was shaken for 30 seconds and the step wasrepeated. The chip was then placed on a petri dish containing cold PBSand shaken for 2 minutes. This was followed by a rinse with DEPC-treatedwater at room temperature for one minute. The chip was then air-driedfor 1 minute.

The chip was placed on a petri dish with Proteinase K at a concentrationof 7.5 μg/mL and incubated at 45° C. for 30 minutes. Once the digestionwas complete, the Proteinase K was denatured by heating the chip at 95°C. for 90 seconds. The chip was washed with PBS for 10 seconds andDEPC-treated water for 30 seconds to remove the residual Proteinase K.

Bulk Loading and On-chip RT-LAMP reactions: A 25 μL reaction mix wasprepared for a single on-chip test, and 10 μL of the reaction mix waspipetted onto the tissue/wells, and immediately coated with a layer ofmineral oil. The chip was then placed in a petri dish covered withmineral oil and degassed for 5 minutes to remove any air bubbles. Afterdegassing, the chip was dipped in mineral oil, and an air pressure isapplied at an angle to shear off and remove excess reagents from top ofthe wells. The chip was then placed on a copper bowl and placed on ahotplate under a fluorescent microscope to perform the on-chip RT-LAMPreactions.

The on-chip reactions were carried on a commercial hotplate at 65° C.for 60 minutes and imaged every 2 minutes under an Olympus BX51fluorescence microscope with 3.6 s exposure settings and 16× gain. TRITCfilter was used for imaging the evagreen fluorescent dye.

Amplification Data Analysis: The off-chip amplification and standardcurves were plotted using a MATLAB script. The threshold time for eachcurve was taken as the time required for each curve to reach 20% of themaximum intensity. For on-chip reactions, the raw fluorescent intensityon-chip was extracted from each well and was plotted against time togenerate the raw fluorescence curves. Each raw amplification curve wasfitted to a sigmoidal curve using a four-point parameter modeling (FIGS.26A-26B). The following equation was used for the analysis:

$f = {y_{0} + \frac{a}{1 + e^{- {(\frac{x - x_{0}}{b})}}}}$

Where f=fluorescence intensity, y₀=background fluorescence at time=0minutes, a=difference between the initial and final fluorescentintensity, x=time point of analysis, x₀=inflection point of the curve,b=slope of the curve. The threshold time was obtained at the point wherethe fluorescent intensity=y₀+0.2*a. The positive and negative wells weredifferentiated on the basis of the R² value of the sigmoidal fit and theparameters a and x₀. The threshold time was taken as the point ofinflection. Negative wells had a combination of low R2 value, low avalue, or a very high threshold time (x₀>50 mins).

TOP2A mRNA FISH Probe Design: FISH probes targeting human TOP2A mRNA(NM_001067.3, 3490 bp to 5753 bp) were designed using Stellaris® ProbeDesigner (version 4.2, LGC Biosearch Technologies, US). Probe sequenceswith 85% or greater homology with mouse TOP2A mRNA complementarysequences were excluded. The resulting set of 36 mRNA FISH probes with3′ Quasar 670 dye label was synthesized by LGC Biosearch Technologies.Probe sequences are listed in Table 3.

Validation of TOP2A mRNA FISH on Cell Lines: Probe specificity wasvalidated on TOP2A-positive human cell lines (LNCaP and PC-3, generousgifts from Dr. Stephen J. Murphy, Mayo Clinic) and mouse cell lines asnegative controls (3T3 and RAW 264.7, purchased from ATCC) followingpreviously published procedures⁶ and protocols provided by the probesupplier. Briefly, 1×10⁵ cells were seeded on 18 mm round #1 coverglassin each well of a 12-well cell culture plate. After adhering, cells werewashed with phosphate buffered saline and fixed with 4% paraformaldehydefor 10 minutes at room temperature. Cells were then permeabilized with70% (v/v) ethanol for 24 hours at 4° C. Ethanol was aspirated and WashBuffer A (LGC Biosearch Technologies) was added. After incubated for 5minutes at room temperature the coverglass was transferred face-downonto Parafilm with 100 μL of Hybridization Buffer (LGC BiosearchTechnologies) containing probes. After incubation for 16 hours in thedark at 37° C. in a sealed humidified chamber, the coverglass was washedwith Wash Buffer A in the dark at 37° C. for 30 minutes. Nuclei werecounterstained with Hoechst 33342 (Thermo Fisher, US) for 30 minutes.The coverglass was then washed with Wash Buffer B (LGC BiosearchTechnologies) for 5 minutes before mounting on slides containingVectashield Mounting Medium, and sealed using nail polish. FISH probesand nuclei were imaged using a Leica SP8 UV/Visible Laser ConfocalMicroscope (Leica, Germany) with a 63× oil-immersion objective.

TOP2A mRNA FISH on Prostate Tumor Tissue: Experimental procedures forFISH staining were reported previously³² and provided by the probesupplier. Tumor tissue sections were stored at −80° C. and equilibratedto room temperature before use. The tissue-mounted slides were immersedin 4% paraformaldehyde fixation buffer for 10 minutes at roomtemperature and then treated in the same way as cell lines for mRNAFISH, with the exception that a 20× oil objective was used for imagecollection.

mRNA FISH and microchip amplification correlation analysis: A pixelatedmRNA FISH image with the same resolution as the spatial threshold mapwas generated by taking the mean fluorescence intensity of theneighbouring pixels. For the correlation analysis, the non-amplifyingpixels were assigned a value of 100 as their threshold time. Thespearman correlation coefficient and the associated p-values between thetwo images were obtained using the corr function in matlab.

TABLE 2 The RT-PCR and RT-LAMP primers were synthesized byIntegrated DNA Technologies (IDT), and are listed below. SEQ ID NO.TOP 2A F3: GTC GTG TCA GAC CTT GAA 1 RT-LAMP primersB3: TAG TTC CTT TTG GGG CAG 2 FIP: TCT GGG AAA TGT GTA GCA GGA GGC TGA 3TGA TGT TAA GGG CA BIP: AAC CCA GTT CCT AAA AAG AAT GTG AGT 4GGA GGT GGA AGA CTG A TOP2A Loop F: GGC TTG AAG ACA GTG GTA CAC 5RT-PCR primers Loop B: CAG TGA AGA AGA CAG CAG CAA 6Forward: TGG CTG CCT CTG AGT CTG AA 7Reverse: AGT CTT CTG CAA TCC AGT CCT CTT 8

Bayesian inference-base method: Metrics were identified by theexamination of spectra, S, by a trained spectroscopist from regionsdelineated by a trained pathologist. From the universal set of metrics,M={m₁, m₂, . . . m_(n)}, an evaluation of pairwise error and incrementalincrease in classification accuracy for every class, C={c₁, c₂, . . . ,c_(i)}, resulted in a subset of 2 metrics. These were further reduced toa set of 18 by leaving out one metric at a time and evaluating theresulting classification accuracy on validation array data. Theclassification process reported here consists of evaluating the maximuma posteriori probability for every class c_(i), p_(i)(c_(i)|M), forevery metric profile from every spectrum from the image M(x,y) toformulate a decision rule

p(c _(j) |M)>p(c _(i) |M), i=1,2, . . . ,n _(c) , i≠j  (1)

Classification evaluation: As the threshold acceptance value determinesan operating point for the algorithm, a systematic variation of theacceptance threshold can be used to carry out validation and statisticalanalyses of the classification results following methods in D CFernandez, R Bhargava, S M Hewitt, I W Levin Nature Biotechnology 23,469-474 (2005).

TABLE 3 mRNA FISH probes for TOP2A mRNA SEQ ID Sequence NO.  1TGTTACGGAGTCACTCTTTT  9  2 AGTTGAAGGTTGGTCCAGAA 10  3ACCAAAGGGGCATATCAAGA 11  4 CCAAGTCTTCTTTCCACAAA 12  5CTTCAACAGCCTCCAATTCT 13  6 TGTTCATCTTGTTTTTCCTT 14  7ATGGTTATTCGTGGAATGAC 15  8 TTTTAGGCCTTCTAGTTCCA 16  9TTGGCTTAAATGCCAATGTA 17 10 GATTCTGAATCAGACCAGGG 18 11AATTACTTTCGTCACTGCTC 19 12 TGTTTCTCGTGGAGGGACAT 20 13TAGGTGGACTAGCATCTGAT 21 14 CTTCAAGGTCTGACACGACA 22 15GGCTTGAAGACAGTGGTACA 23 16 TCTGGGAAATGTGTAGCAGG 24 17ACTGGGTTTGTAATTTCAGT 25 18 AGTGGAGGTGGAAGACTGAC 26 19CAAAGCTGGATCCCTTTTAG 27 20 GCTTTTGAGAGACACCAGAA 28 21ATTCTTGGTTTTGGCAGGAT 29 22 GGATTTCTTGCTTGTGACTG 30 23ATGGAAGTCATCACTCTCCC 31 24 CCACAGCTGAGTCAAAGTCC 32 25TTAAAACCAGTCTTGGGCTT 33 26 TTGGGCTTTACTTCACTTTG 34 27CCATGAGATGGTCACTATTT 35 28 GCTGAAGTGATCAGATAGCT 36 29TGCTCTATCTCATATCTACT 37 30 GAGTATCTGTACTAGAACCA 38 31TTGGCACATAAGAGGCTGAG 39 32 TGAGCAATTTCTCATTGCTT 40 33GGCCTCTGATGATTTGAGAA 41 34 AAATTGGTTTCTCTCTTTGG 42 35CTTGGATCAAATGTTGTCCC 43 36 ATTGCTGAGCATGGTTATCA 44

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EXAMPLE 3 Device for Generating a Pixelized Tissue Sample

In an embodiment, described herein is a device for pixelating a tissuesample. An exemplary device is provided in FIGS. 22A-K and 31A-B. Thedevice includes a plurality of wells 100 supported by or embedded in asubstrate 110 for receiving a tissue sample, including a cryogenichistogram. A shearing surface 120 positioned between the wells has asharp edge to sever a tissue sample 120 when force is applied to it. Adeformable substrate 140 configured to fit over the plurality of wells100 and placed on top of the tissue sample may be used to force tissuesample into the wells and ensure the tissue sample 130 is severed suchthat a portion (e.g., “island”) enters each of the plurality of wells100. When force, such as that provided by a centrifuge, is applied, asindicated by the arrow in FIG. 31B, the tissue sample severs such that aportion is forced into each of the wells generating a tissue sampleisland 150 in each well and thereby creating a pixelized tissue sample.Upon relaxation or removal of the force, the tissue sample remains inthe well, and the deformable substrate relaxes away from the well. Asdesired, the deformable substrate may be removed, with the pixilatedtissue ready for further action, including processing, amplification andimaging, as shown in the right-most panel of FIG. 31B.

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, are disclosedseparately. When a Markush group or other grouping is used herein, suchas compositions, physical dimensions or temperatures, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.Specific names of compounds are intended to be exemplary, as it is knownthat one of ordinary skill in the art can name the same compoundsdifferently.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The methods, chips and kit now will be described more fully hereinafterwith reference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements.

Likewise, many modifications and other embodiments of the methods, chipsand kits described herein will come to mind to one of skill in the artto which the invention pertains having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is to be understood that the invention is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims. Although specific terms are employed herein, they areused in a generic and descriptive sense only and not for purposes oflimitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the embodiments above and the claims below.Reference should therefore be made to the embodiments above and claimsbelow for interpreting the scope of the invention.

1-24. (canceled)
 25. A device for generating a pixelized tissue samplecomprising: a substrate; a plurality of wells supported by or embeddedin said substrate; and a shearing surface positioned between adjacentwells, wherein the shearing surface has a sharp edge configured to severa tissue sample under an applied centrifugal force into a plurality oftissue sample islands, with each well containing a unique tissue sampleisland so as to maintain spatial information of a tissue sample duringuse.
 26. The device of claim 25, wherein said substrate is silicon, aglass, a metal, an insulator or a dielectric.
 27. The device of claim25, wherein said shearing surface is a sharp edge of an invertedpyramidal well.
 28. The device of claim 25, wherein each of said wellscomprises a target-specific primer set and an enzyme for nucleic acidamplification.
 29. The device of claim 25 further comprising adeformable substrate configured to force a tissue sample into each ofsaid plurality of wells and shear the tissue between the shearingsurface and the deformable substrate.
 30. (canceled)
 31. The device ofclaim 25, wherein the substrate comprises undoped silicon.
 32. Thedevice of claim 25 comprising enzymes and/or primers disposed on atleast one surface of the plurality of wells.
 33. The device of claim 25comprising one or more positively charged functional groups disposed onone or more surfaces of the wells.
 34. The device of claim 25 comprisinga silanized layer disposed on one or more surfaces of the wells.
 35. Thedevice of claim 25, wherein each of said wells has a volume of less thanor equal to 1000 pL.
 36. The device of claim 25, wherein each of saidwells has; a cross-sectional dimension of less than or equal to 1 mm,37. The device of claim 25, wherein each of said wells has a maximumdepth of less than or equal to 1 mm.
 38. The device of claim 25, whereinsaid deformable layer comprises a polymer.
 39. The device of claim 38,wherein said polymer comprises polymethylsiloxane (PDMS), SU-8,polyethylene glycol (PEG), a photoresist, a PEG-based polymer or anycombination thereof
 40. The device of claim 25, wherein the microarraycomprises greater than 500 wells.
 41. A system for generating apixelized tissue sample, the system comprising: the device of claim 25;and a centrifuge configured to receive the device and a tissue sampleand shear the tissue sample at the shearing surface.